The neural circuits that govern behaviors vital to mammals, such as locomotion and respiration, rely on the ability of motor neurons (MNs) within the spinal cord to establish selective connections with dedicated sets of peripheral and central synaptic targets. Signaling pathways acting along the dorsoventral axis of the neural tube have been shown to determine the early identity of MNs and distinguish this class from other neuronal types within the spinal cord. The subsequent diversification of MNs depends on the actions of approximately 20 Hox transcription factors, which appear to be required at distinct phases of MN differentiation. While Hox genes are essential for MN fate specification, the targets of their activities are not known, nor is it understood how they achieve MN-specificity, given their relatively broad roles in patterning along the rostrocaudal axis. Moreover the factors that determine the expression patterns of Hox proteins in MNs are poorly defined. In aim1 we will characterize the direct targets of Hox proteins, assess how they are regulated in motor columns, and determine if and how they intersect with MN-specific gene programs. In aim2 we will dissect the mechanisms of Hox protein specificity in controlling facets of MN identity, focusing on the Hoxc9 protein, a central determinant of MN columnar organization. In aim3 we will test the hypothesis that the organization of Hox-dependent MN subtype relies on graded activities of Polycomb proteins that ensure proper postmitotic Hox expression patterns. These studies will provide basic insights into the mechanisms through which Hox proteins influence MN differentiation, and should allow for the design of strategies to generate MN subtypes from undifferentiated cells.